Imaging beta-amyloid peptides and inhibition of beta-amyloid peptide aggregation

ABSTRACT

The present invention relates to methods of detecting and monitoring aggregation of beta-amyloid peptides which are associated with neurodegenerative diseases as well as treating and/or preventing the neurodegenerative diseases by using carbazole-based fluorophores. In particular, the present invention provides methods for labeling and imaging the beta-amyloid (Aβ) peptides, oligomers, and fibrils in vitro and/or in vivo, as well as treating and/or preventing Alzheimer&#39;s disease by using the carbazole-based fluorophores of the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalPatent Application No. 61/477,614 filed Apr. 21, 2011, the disclosure ofwhich is incorporated by reference herein.

FIELD OF INVENTION

The present invention relates to methods of detecting and monitoringaggregation of beta-amyloid peptides which are associated withneurodegenerative diseases as well as treating and/or preventing theneurodegenerative diseases by using carbazole-based fluorophores. Inparticular, the present invention provides methods for labeling andimaging the beta-amyloid (Aβ) peptides, oligomers, and fibrils in vitroand/or in vivo, as well as treating and/or preventing Alzheimer'sdisease by using the carbazole-based fluorophores of the presentinvention.

BACKGROUND OF INVENTION

Loss of memory and cognitive functions are often associated with aging.This is the result of neurodegeneration. However, in some cases, thisprocess of neurodegeneration becomes accelerated due to premature celldeath in the brain, leading to a variety of cognitive impairments ordementia. Among these neurodegenerative disorders, Alzheimer's disease(AD) is most prevalent in recent years. It has also attractedconsiderable attention locally because Prof. Charles K. Kao, formerpresident of the Chinese University of Hong Kong and Nobel Laureate inPhysics, 2009, was stricken with this devastating disease.

More than 26 million people worldwide were estimated to suffer fromAlzheimer's disease (AD) in 2006 and the patient number was expected toincrease by 4-fold in 2050. The incidence rate of AD is known toincrease with age. At age over 65, the incidence rate is about 5% in thegeneral population. At age over 80, the incidence rate increases toabout 20%, i.e., one in five. Current drug treatments can only improvesymptoms and produce no profound cure. In recent years, severalapproaches aimed at inhibiting disease progression have advanced toclinical trials. Among them, strategies targeting the production andclearance of the Aβ peptide, which is thought to be a critical proteininvolved in the pathogenesis of the disease, are the most advanced.

Aβ peptide is the principal protein component of the Aβ plaques whichare found in the brains of AD patients during autopsy. The occurrence ofthe Aβ plaques, considered a cardinal feature of AD, provides the onlyconfirmed diagnosis of the disease. Extensive researches in past decadeshave indicated a central role for the Aβ peptide in the disease processwhere the Aβ peptides assemble (aggregate) into Aβ fibrils which exert acytotoxic effect towards the neurons and initiate the pathogeniccascade. Recent studies showed that oligomeric, prefibrillar anddiffusible assemblies of Aβ peptides are also deleterious. The abilityof this peptide to form Aβ fibrils seems to be largelysequence-independent, and many proteins can form structures with thecharacteristic cross-β stacking perpendicular to the long axis of thefiber. Although a consensus mechanism for the pathogenic oligomericassembly has yet to emerge, the idea of finding some brain-penetratingsmall molecules that can interfere with the interactions among the Aβpeptide monomers and thus inhibit the formation of the neurotoxicoligomers and the resulting Aβ plaques is an attractive approach totreating/preventing the disease. The use of agents that stabilize themonomer, interfere with the aggregation process (amyloidogenesis) andallow for the isolation of the intermediate species will help toelucidate the molecular mechanism of Aβ fibril formation. In addition,imaging agents that can specifically bind Aβ fibrils and plaques invitro and in vivo are of paramount importance for studying thepathological events of the disease, disease diagnosis and monitoring oftherapeutic treatment.

We have previously shown that carbazole-based fluorophores are highlysensitive fluorescent light-up probe for double strand DNA and stronglyactive two-photon absorption dyes for two-photon excited bioimaging(Feng et al, 2010), the disclosure of which is incorporated by referenceherein. Recently, the mono-cyanine fluorophore has also been found toexhibit binding affinity towards beta amyloid (Aβ) peptide concomitantwith strong fluorescent enhancement. These findings provide us the leadmolecular structure to design and synthesize novel functionalcarbazole-based fluorophores for imaging and inhibition the aggregationof Aβ peptides.

Citation or identification of any reference in this section or any othersection of this application shall not be construed as an admission thatsuch reference is available as prior art for the present application.

SUMMARY OF INVENTION

The first aspect of the present invention relates to methods of usingcarbazole-based fluorophores for labelling, imaging and detecting betaamyloid peptides, oligomers, and fibrils that fluoresce strongly uponbinding in vitro or in vivo. The fluorophores of the present inventionare non-toxic and able to cross the blood brain barrier. Thefluorophores could potentially be used as labeling dyes to assist theinvestigation of beta amyloid peptides, oligomers, and fibrils in vitroand in vivo.

Another aspect of the present invention relates to methods of usingcarbazole-based fluorophores for inhibiting the growth of beta amyloidoligomers and preventing their aggregation upon binding in vitro or invivo. The fluorophores of the present invention are non-toxic and ableto cross the blood brain barrier. The fluorophores could potentiallyserve as Alzheimer's disease therapeutics and/or preventive agents.

A further aspect of the present invention uses carbazole-basedfluorophores that bind beta amyloid peptides as a magnetic resonanceimaging (MRI) contrast agent. By conjugating appropriate paramagneticmetal complexes to these carbazole-based fluorophores, these compoundscan potentially be developed into beta-amyloid peptide-specific MRIcontrast agents.

General

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described.

The invention includes all such variation and modifications. Theinvention also includes all of the steps and features referred to orindicated in the specification, individually or collectively, and anyand all combinations or any two or more of the steps or features.

Throughout this specification, unless the context requires otherwise,the word “comprise” or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated integer or groupof integers but not the exclusion of any other integer or group ofintegers. It is also noted that in this disclosure and particularly inthe claims and/or paragraphs, terms such as “comprises”, “comprised”,“comprising” and the like can have the meaning attributed to it in U.S.Patent law; e.g., they can mean “includes”, “included”, “including”, andthe like; and that terms such as “consisting essentially of” and“consists essentially of” have the meaning ascribed to them in U.S.Patent law, e.g., they allow for elements not explicitly recited, butexclude elements that are found in the prior art or that affect a basicor novel characteristic of the invention.

Furthermore, throughout the specification and claims, unless the contextrequires otherwise, the word “include” or variations such as “includes”or “including”, will be understood to imply the inclusion of a statedinteger or group of integers but not the exclusion of any other integeror group of integers.

Other definitions for selected terms used herein may be found within thedetailed description of the invention and apply throughout. Unlessotherwise defined, all other technical terms used herein have the samemeaning as commonly understood to one of ordinary skill in the art towhich the invention belongs.

Other aspects and advantages of the invention will be apparent to thoseskilled in the art from a review of the ensuing description.

BRIEF DESCRIPTION OF INVENTION

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The above and other objects and features of the present invention willbecome apparent from the following description of the invention, whentaken in conjunction with the accompanying drawings, in which:

FIG. 1 shows the fluorescence spectra of SPM, SPOH, SLM, SLOH, SLE,SLOH-Pr, Me-SLM, SAM, and SAOH (1 μM) in phosphate buffer upon additionof various concentrations of Aβ(1-40) fibrils prepared from Aβ₄₀ withseed incubated at 37° C. for an hour in buffer (left column). FIG. 1Ashows the fluorescence spectra of SPM in various concentrations ofAβ(1-40) fibrils. FIG. 1C shows the fluorescence spectra of SPOH invarious concentrations of Aβ(1-40) fibrils. FIG. 1E shows thefluorescence spectra of SLM in various concentrations of Aβ(1-40)fibrils. FIG. 1G shows the fluorescence spectra of SLOH in variousconcentrations of Aβ(1-40) fibrils. FIG. 1I shows the fluorescencespectra of SLE in various concentrations of Aβ(1-40) fibrils. FIG. 1Kshows the fluorescence spectra of SLOH-Pr in various concentrations ofAβ(1-40) fibrils. FIG. 1M shows the fluorescence spectra of Me-SLM invarious concentrations of Aβ(1-40) fibrils. FIG. 1O shows thefluorescence spectra of SAM in various concentrations of Aβ(1-40)fibrils. FIG. 1Q shows the fluorescence spectra of SAOH in variousconcentrations of Aβ(1-40) fibrils. FIG. 1 also shows the fluorescencespectra of SPM, SPOH, SLM, SLOH, SLE, SLOH-Pr, Me-SLM, SAM, and SAOH (1μM) in phosphate buffer itself, in the presence of 100 equv of Aβ₄₀ inmonomeric form, and in the presence of 100 equv of Aβ₄₀ in fibril statein phosphate buffer (right column). FIG. 1B shows the fluorescencespectra of SPM on its own and in the presence of two different forms ofAβ₄₀. FIG. 1D shows the fluorescence spectra of SPOH on its own and inthe presence of two different forms of Aβ₄₀. FIG. 1F shows thefluorescence spectra of SLM on its own and in the presence of twodifferent forms of Aβ₄₀. FIG. 1H shows the fluorescence spectra of SLOHon its own and in the presence of two different forms of Aβ₄₀. FIG. 1Jshows the fluorescence spectra of SLE on its own and in the presence oftwo different forms of Aβ₄₀. FIG. 1L shows the fluorescence spectra ofSLOH-Pr on its own and in the presence of two different forms of Aβ₄₀.FIG. 1N shows the fluorescence spectra of Me-SLM on its own and in thepresence of two different forms of Aβ₄₀. FIG. 1P shows the fluorescencespectra of SAM on its own and in the presence of two different forms ofAβ₄₀. FIG. 1R shows the fluorescence spectra of SAOH on its own and inthe presence of two different forms of Aβ₄₀.

FIG. 2 shows the fluorescence spectra of SPM, SPOH, SLM and SLOH inphosphate buffer (1 μM) upon addition of various concentrations ofAβ(1-40) (left column) and Aβ(1-42) (right column), respectively. FIG.2A shows the fluorescence spectra of SPM in various concentrations ofAβ(1-40). FIG. 2B shows the fluorescence spectra of SPM in variousconcentrations of Aβ(1-42). FIG. 2C shows the fluorescence spectra ofSPOH in various concentrations of Aβ(1-40). FIG. 2D shows thefluorescence spectra of SPOH in various concentrations of Aβ(1-42). FIG.2E shows the fluorescence spectra of SLM in various concentrations ofAβ(1-40). FIG. 2F shows the fluorescence spectra of SLM in variousconcentrations of Aβ(1-42). FIG. 2G shows the fluorescence spectra ofSLOH in various concentrations of Aβ(1-40). FIG. 2H shows thefluorescence spectra of SLOH in various concentrations of Aβ(1-42).

FIG. 3 shows TIRFM images of Aβ fibrils after incubation with thecarbazole-based fluorophores, SPM (left) excited at 445 nm and SLM(middle) and SLOH (right) excited at 488 nm, respectively.

FIG. 4 shows in vitro fluorescence imaging of neuronal cells by usingthe carbazole-based fluorophore, SLOH. (left) The lambda scans of theimages match well with the fluorescence spectrum of the SLOH (right).

FIG. 5 shows absorption and fluorescence spectra of the carbazole-basedfluorophores, SAM (left) and SAOH (right) in phosphate buffer solution.

FIG. 6 shows CD spectra of Aβ(1-40) peptide (upper) and fibrils (lower)in the absence and presence of SLOH (1:1) (20 μM).

FIG. 7 shows TIRFM images of Aβ fibrils (left) and Aβ peptide afterincubation with the carbazole-based fluorophore, SLOH (upper middle),SAOH (upper right), SLE (lower left), SLOH-Pr (lower middle), and Me-SLM(lower right). The middle and right panels show an inhibition of Aβfibril formation from the Aβ monomer by SLOH and SAOH. These images wereobtained by an addition of ThT dye excited at 445 nm.

FIG. 8 shows TEM images of Aβ fibril growth from Aβ peptide (50 μM)seeded for 1 hr at 37° C. in the absence (right panel) and the presenceof SLOH (left panel).

FIG. 9 shows ThT, SLM and SLOH fluorescence binding assays for 50 μMAβ₄₀ fibrillation (left panel). Average length of 1 h incubatedAβ40-fibril measured from TIRFM images after 1 h seed-mediatedincubation of Aβ40 monomer with (bottom axis, bars) and without (topaxis, scatter point) SLOH (50 μM) added at different time points (0, 10,20, 40 and 60 min) within an one hour-incubation. (right panel).

FIG. 10 shows cytotoxicities of the carbazole-based SLOH, SLOH-Pr, andMe-SLM towards the SH-SY5Y neuronal cell with MTT assays.

FIG. 11 shows cytotoxicities of Aβ peptide monomer (left), oligomers(center) and fibrils (right) towards the SH-SY5Y neuronal cell in theabsence and the presence of SLOH (50 μM) (upper left), SLE (upperright), SLOH-Pr (lower left), Me-SLM (lower right) after 2 hr, 6 hr, and24 hr incubations.

FIG. 12 shows fluorescence images of transgenic mice brain with tailvein injection of SLOH and co-stained with the Aβ labeling dye, ThT orAβ antibody with DAB stain. Fluorescence image corresponding to SLOHfluorescence captured at 550-630 nm under excitation at 488 nm (upperleft); ThT fluorescence captured at 470-550 nm under excitation at 458nm (upper middle); and overlapped images of previous two images (upperright). The overlapped image revealed the colocalization of fluorescencesignals of SLOH and ThT in cellular components. DifferentialInterference Contrast (DIC) image of DAB stained brain slide oftransgenic mice (lower left). Fluorescence image of same slidecorresponding to SLOH fluorescence captured at 550-630 nm underexcitation at 488 nm (lower middle); and overlapped images of previoustwo images (lower left). The overlapped image revealed thecolocalization of fluorescence signals of SLOH and Aβ antibody incellular components.

FIG. 13 shows synthesis of carbazole-based fluorophores, SPM, SPOH, SLM,SLOH, SLE, SLOH-Pr, Me-SLM, SAM, and SAOH.

DETAILED DESCRIPTION OF INVENTION

The present invention is not to be limited in scope by any of thespecific embodiments described herein. The following embodiments arepresented for exemplification only.

The general chemical structures of carbazole-based fluorophores,including S or V series, are shown as follows:

wherein Ar is a heteraromatic ring selected from the group consisting ofpyridinyl, substituted pyridinyl, quinolinyl, substituted quinolinyl,acridinyl, substituted acridinyl, benzothiazolyl, substitutedbenzothiazolyl, benzoxazolyl, and substituted benzoxazolyl; R₁ is aradical selected from the group consisting of polyethylene glycol chain,alkyl, substituted alkyl, peptide chain, glycosidyl, andC(O)NHCH((CH₂CH₂O)₂CH₃)₂; R₂ is selected from the group consisting ofethenyl, ethynyl, azo and azomethinyl; R₃ is a radical selected from thegroup consisting of alkyl, HO-alkyl, HS-alkyl, H₂N-alkyl, HNalkyl-alkyl,HOOC-alkyl, (alkyl)₃N⁺-alkyl, and (Ph)₃P⁺-alkyl; X is an anion selectedfrom the group consisting of F, Cl, Br, I, HSO₄, H₂PO₄, HCO₃, tosylate,and mesylate.

In one embodiment, Ar is a quinolinyl or substituted quinolinyl; R₁ is a2-(2-methoxyethoxy)ethoxy; R₂ is an ethenyl; R₃ is a methyl,2-hydroxyethyl, ethyl or 3-hydroxypropyl; and X is a chloride, bromideor iodide, and the compounds of which are represented by the aboveformula “SLM”, “SLOH”, “SLE” and “SLOH-Pr”, respectively.

In another embodiment, Ar is a quinolinyl or substituted quinolinyl; R₁is a methyl; R₂ is an ethenyl; R₃ is a methyl; and X is a chloride,bromide or iodide, the compounds of which are represented by the aboveformula Me-SLM.

In a further embodiment, Ar is an acridinyl or substituted acridinyl; R₁is a 2-(2-methoxy-ethoxy)ethoxy; R₂ is an ethenyl; R₃ is a methyl or2-hydroxyethyl; and X is selected from a chloride, bromide or iodide,and the fluorophores of which are represented by the above formula SAMand SAOH, respectively, where the difference between the compounds ofSAM and SAOH is the substitutent at R₃.

In other embodiment, Ar is selected from a pyridinyl or substitutedpyridinyl, R₁ is a 2-(2-methoxyethoxy)ethoxy; R₂ is an ethenyl; R₃ isselected from a methyl or 2-hydroxyethyl; and X is selected from achloride, bromide or iodide, the compounds of which are represented bythe formula SPM and SPOH, respectively.

A novel series of water-soluble carbazole-based fluorophores has beendesigned and developed. These molecules were found to bind to Aβ(1-40)and Aβ(1-42) peptides and, more specifically, their oligomers, andfibrils with strong fluorescence enhancement, therefore allowing directimaging and detection for the Aβ peptides, oligomers and their fibrils(FIG. 1). Upon binding with Aβ peptides, there is about 8- to about82-fold increase in fluorescence intensity concomitant with thesubstantial blue shifts (Δ=14-22 nm) in the emission spectra of thefluorophores (FIG. 2). Interestingly, the fluorescence enhancement ismuch stronger for fibrils than peptides. (e.g. F_(fibril)/F_(SLOH)=81.5vs. F_(peptide)/F_(SLOH)=6.3). Because of such strong increase influorescence, the signal-to-noise ratio is so high that imaging ofsingle fibrils is possible. (FIG. 3) Compared to common commerciallabeling dyes for Aβ such as Thioflavin-T and Congo Red, thecarbazole-based fluorophores of the present invention provide anadvantage of a wide range of excitation and emission tuning in visibleto infra-red region (FIG. 4). Some of these molecules, e.g., SAM andSAOH, even emit at ˜760 nm (FIG. 5), which can potentially be used fornear infra-red fluorescence imaging. In addition to fluorescencetitration, the binding of Aβ peptide and fibril with the carbazole-basedfluorophores of the present invention were further confirmed by circulardichroism spectroscopy (FIG. 6), and electrospray ionization-massspectrometry (ESI-MS). Total Internal Reflection Fluorescence Microscope(TIRFM) technique developed by us was used to investigate the inhibitioneffects of these functional fluorophores on Aβ fibril formation (FIG.7). Remarkably, some of these molecules, e.g., SLOH, SLE, SLOH-Pr,Me-SLM, SAM, and SAOH, were found to inhibit Aβ peptide aggregation andprevent fibril growth (FIG. 7). Such inhibitory effect was furtherconfirmed by Transmission Electron Microscopy (TEM) study (FIG. 8).

The inhibitory effect of the carbazole-based fluorophores of the presentinvention on Aβ fibril growth was further investigated by measuring the(average) length of the Aβ fibrils formed after incubation of the Aβmonomers for 60 min with additions of SLOH at different time pointsduring this period (FIG. 9). Parallel experiments conducted without anyaddition of SLOH were used as controls. FIG. 9 shows that an addition ofSLOH to the Aβ monomer strongly arrests its fibril growth. These resultsclearly indicate that the inhibitory effect of these carbazole-basedfluorophores on Aβ aggregation is instantaneous.

To ascertain its potential clinical application, the cytotoxicities ofthese carbazole-based molecules, SLOH, SLOH-Pr, Me-SLM, and SAOH towardsthe neuronal cell, i.e., SH-SY5Y cell line, were investigated by MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reductionassay. The results obtained (FIG. 10) showed that these molecules wereessentially non-toxic (20%) to the neuronal cell particularly at lowdosage.

Since it is the Aβ oligomers and fibrils that are neurotoxic, furtherexperiments with these carbazole-based molecules conducted in thepresence of the Aβ monomer (non-toxic), the neurotoxic Aβ oligomers andfibrils showed that the neuronal cells became protected from theneurotoxic effects of the Aβ oligomers and fibrils when incubated withcarbazole-based molecules SLOH and SAOH for 2 and 6 hours (FIG. 11).

However, in order for the observed neuroprotective effect to beclinically useful, these molecules need to be able to pass through theblood-brain barrier. The ability of these molecules to penetrate theblood-brain barrier was demonstrated in transgenic mice (FIG. 12). Inaddition, FIG. 12 d-f shows the selectivity of SLOH towards Aβ plaquesas confirmed with Aβ antibody which was used to identify the Aβ plaquesin transgenic mice's brain.

In summary, carbazole-based fluorophores of the present invention havebeen shown to bind to Aβ₍₁₋₄₀₎ and Aβ₍₁₋₄₂₎ as well as Aβ aggregateswith dramatic fluorescence enhancement, thus allowing their directimaging and labeling as well as the use of TIRFM technique to study theeffects of these molecules on Aβ aggregation/fibrillation. Someembodiments of the carbazole-based fluorophores, for instance, SLOH andSAOH, have been shown to be a potent inhibitor of Aβ aggregation,non-toxic and exhibiting a protective effect against the neurotoxicactivities of the Aβ oligomers and fibrils towards neuronal cells. Theseproperties, together with the ability to cross the blood-brain barrierand target the Aβ plaques, render the fluorophores of the presentinvention a potential neuroprotective and, perhaps, therapeutic agentfor Alzheimer's disease.

The following compositions according to the invention were prepared andexemplified as shown in FIG. 13. By adapting the convergent approachestablished previously, the Knoevenagel reaction ofcarbazolyl-3-aldehyde and the corresponding 4-methylpyridium or4-methylquinolinium halide was used as the key step to synthesizevarious carbazole-based cyanines. Alkylation of carbazole with ethyleneglycol chloride and methyl iodide in the presence of NaH in DMF gavealkylated carbazole 1a and 1b respectively. Monobromination of 1a and 1bin the presence of NBS gave alkylated 3-bromocarbazole, 2a and 2b,respectively. Formylation of 2a and 2b via lithiation bromide exchangeat low temperature followed by the subsequent quenching with DMFafforded carbazolyl-3-aldehyde, 3a and 3b, respectively, in moderateyield. Alkylation of lepidine or picoline was carried out in methanol oracetonitrile affording the corresponding halide, 4-9 in good to highyield. The Knoevenagel reaction of aldehyde 3a or 3b and thecorresponding 4-methylpyridium or 4-methylquinolinium halide in thepresence of piperidine in ethanol afforded the correspondingcarbazole-based cyanines in a moderate yield. For the acridine-basedcyanines dyes, 9-methylacridine was first brominated with NBS affordingbrominated product 10, which gave phosphonate ester 11 by refluxing withtriethyl phosphite. Condensation of phosphonate 11 and aldehyde 3a inthe presence of NaH afforded 12, which was alkylated with methyl iodideand 2-iodoethanol to give SAM and SAOH, respectively. All the cyanineswere fully characterized with spectroscopic techniques and found to bein good agreement with its structure.

All the solvents were dried by the standard methods wherever needed. ¹HNMR spectra were recorded using a Bruker-400 NMR spectrometer andreferenced to the residue CHCl₃ 7.26 ppm or DMSO-d₆ 2.5 ppm. ¹³C NMRspectra were recorded using a Bruker-400 NMR spectrometer and referencedto the CDCl₃ 77 ppm or DMSO-d₆ 39.5 ppm. Mass Spectroscopy (MS)measurements were carried out by using fast atom bombardment on the APIASTER Pulser I Hybrid Mass Spectrometer or matrix-assisted laserdesorption ionization-time-of-flight (MALDI-TOF) technique. Elementalanalysis was carried on the CARLO ERBA 1106 Elemental Analyzer. Compound8 and SPM were synthesized according to previous procedure.

Apart from the use in direct imaging or labeling of Aβ aggregates, thecarbazole-based fluorophores of the present invention is also useful asa magnetic resonance imaging (MRI) contrast agent that bind beta amyloidpeptides. By conjugating appropriate paramagnetic metal complexes tothese carbazole-based fluorophores, these compounds can potentially bedeveloped into beta-amyloid peptide-specific MRI contrast agents. Toconvert these Aβ fibril-specific carbazole-based fluorophores dyes intoMRI contrast agents, we can attach strongly paramagnetic and kineticallyinert metal complexes, such as the gadolinium(III), iron(III), andmanganese(II) complexes, via the R₁ side chain of the carbazole moietyto these fluorophores. Gd(III)-based chelates, such as [Gd(DTPA)(H₂O)]²⁻(DTPA=diethylenetriaminepentaacetic acid), approved for clinical use in1988 and commercially known as Magnevist, are attractive candidates.Recently, further enhancement of the MRI contrast properties of theseGd(III) complexes was achieved by allowing the coordination of a secondinner-sphere water molecule, which raised the relaxivity of theconventional Gd(III) contrast agents from 4-5 mM⁻¹s⁻¹ (at 20 MHz fieldstrength) to 10.5 mM⁻¹s⁻¹, in the Gd-TREN-1-Me-3,2-HOPO complex, [1](where TREN=tris(2-aminoethyl), HOPO=hydroxypyridinone, structure shownbelow).

A slight modification of one of the hydroxypyridinone ligands of theGd(III) complex, shown in [2], allows flexible attachment to thecarbazole moiety of A fibril-specific dyes via, for example, apolyethylene glycol (PEG) linkage.

More recently, Parker and coworkers (2010) have designed a series of¹H/¹⁹F dual MR imaging agents based on CF₃-labeled lanthanide(III)complexes (Ln=Gd, Tb, Dy, Ho, Er, Tm) with amide-substituted1,4,7,10-tetraazacyclododecane ligand. An example of this ligand systembearing a CF₃ reporter group is shown in [3].

The advantage of ¹⁹F MRI is the exquisite sensitivity of the ¹⁹F shiftof the reporter group to its local chemical environment, thus opening upthe possibility of responsive MRI to detect changes in local pH, oxygenstress, etc. The fact that standard MRI instruments can be easily tunedfrom ¹H to ¹⁹F nuclei, which have very similar magnetic properties, isan added bonus of this technique. This ligand system is also amenable tocoupling (e.g., at the −X or −Y positions indicated) to the carbazolemoiety of the carbazole-based fluorophores dyes.

SYNTHESIS EXAMPLES 9-(2-(2-methoxyethoxy)ethyl)-9H-carbazole (1a)

To a solution of carbazole (3.34 g, 20 mmol) in DMF (80 mL) at 0° C. wasadded NaH (0.72 g, 30 mmol). After heating to 80° C. for 1.5 h,1-chloro-2-(2-methoxyethoxy)ethane (3.31 g, 24 mmol) was added dropwise.The resulting mixture was kept at 80° C. overnight. After cooling downto 0° C., the reaction mixture was carefully quenched with water andextracted with ethyl acetate three times. The combined organic phase waswashed with water and brine. Then the organic layer was dried overanhydrous sodium sulfate and the solvent was removed. The residue waspurified by silica gel chromatography using petroleum ether and ethylacetate as eluent (EA:PE=1:3) to afford alkylated carbazole 1a (4.46 g)as brown oil in 83% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.09 (d, J=7.6 Hz,2H), 7.46 (m, 4H), 7.23 (m, 2H), 4.51 (t, J=6.4 Hz, 2H), 3.86 (t, J=6.4Hz, 2H), 3.52 (m, 2H), 3.42 (m, 2H), 3.31 (s, 3H). ¹³C NMR (400 MHz,CDCl₃) δ 140.5, 125.6, 122.8, 120.2, 118.9, 108.7, 71.8, 70.7, 69.1,59.0, 43.0. MS (FAB) m/z Calcd for C₁₇H₁₉NO₂ 269.1. Found 269.2 [M]⁺.

9-methyl-9H-carbazole (1b)

To a solution of carbazole (3.34 g, 20 mmol) in DMF (80 mL) at 0° C. wasadded NaH (0.72 g, 30 mmol). After heating at 80° C. for 1.5 h,iodomethane (3.4 g, 24 mmol) was added dropwise. The resulting mixturewas kept at 80° C. overnight. After cooling down to 0° C., the reactionmixture was carefully quenched with water and extracted with ethylacetate three times. The combined organic phase was washed with waterand brine. Then the organic layer was dried over anhydrous sodiumsulfate and the solvent was removed. The residue was purified by silicagel chromatography using petroleum ether and ethyl acetate as eluent(EA:PE=1:5) to afford methylated carbazole 1b (2.78 g) as yellow oil in77% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.08 (d, J=8.0 Hz, 2H), 7.46 (t,J=8.0 Hz, 2H), 7.36 (d, J=8.0 Hz, 2H), 7.22 (t, J=8.0 Hz, 2H), 3.79 (s,3H). ¹³C NMR (400 MHz, CDCl₃) δ 140.9, 125.6, 122.7, 120.2, 118.8,108.4, 28.9.

3-bromo-9-(2-(2-methoxyethoxy)ethyl)-9H-carbazole (2a)

To a solution of 1a (2 g, 7.4 mmol) in dichloromethane (60 mL) was addedNBS (1.3 g, 7.4 mmol) portionwise in an ice-water bath. After completeaddition, the solution mixture was warmed to room temperature andstirred overnight. The resulting solution was washed with water andbrine. The organic phase was dried over anhydrous sodium sulfate and thesolvent was removed. The residue was purified by silica gelchromatography using ethyl acetate and petroleum ether (EA:PE=1:5) aseluent to afford 2a (1.75 g) in 68% yield as an oil that can turn intosolid after standing. NMR (400 MHz, CDCl₃) δ 8.16 (d, J=2.0 Hz, 1H),8.01 (d, J=8.0 Hz, 1H), 7.51 (dd, J=8.0 Hz, 2.0 Hz, 1H), 7.44 (m, 2H),7.34 (d, J=8.4 Hz, 1H), 7.22 (m, 1H), 4.46 (t, J=6.0 Hz, 2H), 3.83 (t,J=6.0 Hz, 2H), 3.48 (m, 2H), 3.39 (m, 2H), 3.28 (s, 3H). ¹³C NMR (400MHz, CDCl₃) δ 140.7, 139.2, 128.2, 126.3, 124.5, 122.8, 121.8, 120.4,119.3, 111.7, 110.4, 109.0, 71.8, 70.7, 69.1, 59.0, 43.2. MS (FAB) m/zCalcd for C₁₇H₁₈BrNO₂ 347.0. Found 347.3 [M]⁺.

3-bromo-9-methyl-9H-carbazole (2b)

To a solution of 1b (2.5 g, 13.8 mmol) in dichloromethane (80 mL) wasadded NBS (2.4 g, 13.8 mmol) portion-wise in an ice-water bath. Aftercomplete addition, the solution mixture was warmed to room temperatureand stirred overnight. The resulting solution was washed with water andbrine. The organic phase was dried over anhydrous sodium sulfate and thesolvent was removed. The residue was purified by silica gelchromatography using ethyl acetate and petroleum ether (EA:PE=1:10) aseluent to afford 2b (2.11 g) in 59% yield. ¹H NMR (400 MHz, CDCl₃) δ8.19 (d, J=2.0 Hz, 1H), 8.03 (d, J=8.0 Hz, 1H), 7.54 (dd, J=8.8 Hz,J=2.0 Hz, 1H), 7.50 (td, J=8.0 Hz, J=1.2 Hz, 1H), 7.39 (d, J=8.0 Hz,1H), 7.27-7.22 (m, 2H), 3.82 (s, 3H).

9-(2-(2-methoxyethoxy)ethyl)-9H-carbazole-3-carbaldehyde (3a)

To a solution of 2a (1.5 g, 4.3 mmol) in dried THF (45 mL) was addedn-BuLi (3.5 mL 5.2 mmol) at −78° C. The resulting mixture was stirred at−78° C. for 1 h and then added with dried DMF (3 mL). The reactionmixture was allowed to warm to room temperature and stirred overnightbefore quenched with aqueous ammonia chloride solution. Water was addedand extracted with ethyl acetate three times. The combined organic phasewas washed with brine and dried over anhydrous sodium sulfate. Afterremoving the solvent, the residue was purified by silica gelchromatography using ethyl acetate and petroleum ether (EA:PE=1:2) aseluent to afford 3a (0.76 g) as yellow solid in 60% yield. NMR (400 MHz,CDCl₃) δ 10.07 (s, 1H), 8.58 (d, J=0.8 Hz, 1H), 8.13 (d, J=8.0 Hz, 1H),7.98 (dd, J=8.8 Hz, 0.8 Hz, 1H), 7.51 (m, 3H), 7.30 (m, 1H), 4.53 (t,J=6.0 Hz, 2H), 3.87 (t, J=6.0 Hz, 2H), 3.49 (m, 2H), 3.38 (m, 2H), 3.26(s, 3H). ¹³C NMR (400 MHz, CDCl₃) δ 191.8, 144.3, 141.1, 128.5, 127.1,126.6, 123.7, 123.0, 122.9, 120.6, 120.4, 109.4, 109.3, 71.8, 70.8,69.1, 59.0, 43.4. MS (FAB) m/z Calcd for C₁₈H₁₉NO₃ 297.1. Found 297.3[M]⁺.

9-methyl-9H-carbazole-3-carbaldehyde (3b)

To a solution of 2b (1.8 g, 6.9 mmol) in dried THF (45 mL) was addedn-BuLi (3.3 mL 8.3 mmol) at −78° C. The resulting mixture was stirred at−78° C. for 1 h and then added with dried DMF (8 mL). The reactionmixture was allowed to warm to room temperature and stirred overnightbefore quenched with aqueous ammonia chloride solution. Water was addedand extracted with ethyl acetate three times. The combined organic phasewas washed with brine and dried over anhydrous sodium sulfate. Afterremoving the solvent, the residue was purified by silica gelchromatography using ethyl acetate and petroleum ether (EA:PE=1:4) aseluent to afford 3b (0.86 g) in 60% yield. ¹H NMR (400 MHz, CDCl₃) δ9.58 (s, 1H), 7.79 (s, 1H), 7.49 (d, J=7.6 Hz, 1H), 7.41 (d, J=8.8 Hz,1H), 7.09 (t, J=7.6 Hz,), 6.90 (t, J=7.6 Hz, 1H), 6.77 (d, J=8.0 Hz,1H), 6.61 (d, J=8.4 Hz, 1H), 3.00 (s, 3H). ¹³C NMR (400 MHz, CDCl₃) δ190.9, 143.2, 140.5, 127.4, 125.8, 122.7, 121.7, 119.6, 119.4, 108.3,107.6, 28.0.

1,4-dimethylquinolinium iodide (4)

A solution mixture of lepidine (0.66 g, 4.65 mmol) and iodomethane (1.32g, 9.3 mmol) in methanol (30 mL) was heated to reflux in a sealed tubeovernight. After cooling to room temperature, methanol was removed undervacuum. Anhydrous acetone was added to the residue and filtered. Theresulting solid was washed with acetone and dried to afford iodide 4(1.1 g) as yellow solid in 83% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 9.35(d, J=6 Hz, 1H), 8.54 (d, J=8.8 Hz, 1H), 8.49 (d, J=8.8 Hz, 1H), 8.27(t, J=7.2 Hz, 1H), 8.07 (t, J=4.8 Hz, 1H), 8.05 (d, J=6 Hz, 1H), 4.57(s, 3H), 3.00 (s, 3H). ¹³C NMR (400 MHz, DMSO-d₆) δ 158.1, 148.9, 137.6,134.9, 129.6, 128.4, 126.8, 122.4, 119.5, 44.9, 19.6. MS (FAB) m/z Calcdfor C₁₁H₁₂N⁺ 158.0. Found 158.2 [M]⁺.

1-(2-hydroxyethyl)-4-methylquinolinium chloride (5)

A solution mixture of lepidine (0.8 g, 5.6 mmol) and 2-chloroethanol(2.25 g, 28 mmol) in acetonitrile (15 mL) was heated to 120° C. in asealed tube overnight. After cooling to room temperature, the solventwas removed. The resulting mixture was precipitate from methanol andethyl acetate to give the desired product 5 (0.79 g) in 63% yield. ¹HNMR (400 MHz, DMSO-d₆) δ 9.24 (d, J=6 Hz, 1H), 8.61 (d, J=7.2 Hz, 1H),8.55 (d, J=7.2 Hz, 1H), 8.25 (m, 1H), 8.06 (m, 2H), 5.15 (br, 1H), 5.08(t, J=4.8 Hz, 2H), 3.91 (t, J=4.8 Hz, 2H), 3.01 (s, 3H). ¹³C NMR (400MHz, DMSO-d₆) δ 158.8, 149.2, 137.1, 135.1, 129.7, 129.1, 127.2, 122.4,119.5, 59.4, 59.0, 19.9. MS (FAB) m/z Calcd for C₁₂H₁₄NO⁺ 188.2. Found188.2 [M]⁺.

1-ethyl-4-methylquinolinium bromide (6)

A solution mixture of lepidine (0.5 g, 3.5 mmol) and bromoethane (1.96g, 18 mmol) in acetonitrile (15 mL) was heated to reflux overnight.After cooling to room temperature, the solvent was removed. Theresulting mixture was precipitate from methanol and ethyl acetate togive the desired product 6 (0.81 g) in 92% yield. ¹H NMR (400 MHz,DMSO-d₆) δ 9.44 (d, J=6 Hz, 1H), 8.60 (d, J=9.2 Hz, 1H), 8.54 (dd, J=8.4Hz, J=1.2 Hz, 1H), 8.26 (td, J=8.0 Hz, J=1.6 Hz, 1H), 8.09-8.04 (m, 2H),5.06 (tr, J=7.2 Hz, 2H), 3.00 (s, 3H), 1.58 (t, J=7.2 Hz, 3H). ¹³C NMR(400 MHz, DMSO-d₆) δ 158.4, 148.2, 136.6, 135.1, 129.6, 128.9, 127.2,122.8, 119.2, 52.5, 19.7, 15.2.

1-(3-hydroxypropyl)-4-methylquinolinium bromide (7)

A solution mixture of lepidine (0.5 g, 3.5 mmol) and 3-bromopropanol(1.9 g, 14 mmol) in acetonitrile (15 mL) was heated to reflux overnight.After cooling to room temperature, the solvent was removed. Theresulting mixture was precipitate from methanol and ethyl acetate togive the desired product 7 (0.83 g) in 84% yield. ¹H NMR (400 MHz,DMSO-d₆) δ 9.41 (d, J=6 Hz, 1H), 8.58 (d, J=8.8 Hz, 1H), 8.54 (dd, J=8.8Hz, J=1.2 Hz, 1H), 8.26 (td, J=8.0 Hz, J=1.2 Hz, 1H), 8.08-8.03 (m, 2H),5.09 (t, J=6.8 Hz, 2H), 3.52 (t, J=5.6 Hz, 2H), 3.01 (s, 3H), 2.15-2.08(m, 2H). ¹³C NMR (400 MHz, DMSO-d₆) δ 158.5, 148.8, 136.8, 135.1, 129.5,128.9, 127.2, 122.6, 119.3, 57.4, 54.8, 32.0, 19.7.

1-(2-hydroxyethyl)-4-methylpyridinium chloride (9)

A solution mixture of picoline (0.93 g, 10 mmol) and 2-chloroethanol(4.03 g, 50 mmol) in acetonitrile (20 mL) was heated to 120° C. in asealed tube overnight. After cooling to room temperature, the solventwas removed under vacuum. The resulting mixture was precipitate frommethanol and ethyl acetate to give the desired product 9 (1.5 g) in 87%yield. NMR (400 MHz, DMSO-d₆) δ 8.94 (d, J=6.4 Hz, 2H), 7.98 (d, J=6.4Hz, 2H), 5.55 (br, 1H), 4.64 (t, J=4.8 Hz, 2H), 3.81 (t, J=4.8 Hz, 2H),2.60 (s, 3H). ¹³C NMR (400 MHz, DMSO-d₆) δ 158.7, 144.2, 127.9, 62.1,60.0, 21.4.

(E)-1-(2-hydroxyethyl)-4-(2-(9-(2-(2-methoxyethoxy)ethyl)-9H-carbazol-3-yl)vinyl)pyridiniumchloride (SPOH)

A solution mixture of 3a (0.13 g, 0.75 mmol), 9 (0.27 g, 0.9 mmol) andpiperidine (0.1 mL) in ethanol (30 mL) was heated to reflux overnight.After cooling down to room temperature, the organic solvent was removedby rotary evaporation. The residue was purified by recrystallizationfrom methanol affording SPOH (0.18 g) as pale red solid in 53% yield.NMR (400 MHz, DMSO-d₆) δ 8.88 (d, J=6.8 Hz, 2H), 8.55 (s, 1H), 8.19 (m,4H), 7.84 (d, J=8 Hz, 1H), 7.65 (m, 2H), 7.49 (m, 2H), 7.25 (t, J=7.2Hz, 1H), 5.66 (s, 1H), 4.57 (m, 4H), 3.79 (m, 4H), 3.43 (m, 2H), 3.27(m, 2H), 3.08 (s, 3H). ¹³C NMR (400 MHz, DMSO-d₆) δ 153.4, 144.4, 142.4,141.7, 140.8, 126.4, 126.3, 126.2, 122.7, 122.6, 122.1, 121.1, 120.3,120.0, 119.7, 110.4, 110.2, 71.2, 69.8, 68.8, 61.6, 600.1, 58.1, 42.8.HRMS (MALDI-TOF) m/z Calcd for C₂₆H₂₉N₂O₃ 417.2172. Found 417.2184 [M⁺].

(E)-4-(2-(9-(2-(2-methoxyethoxy)ethyl)-9H-carbazol-3-yl)vinyl)-1-methylquinoliniumiodide (SLM)

A solution mixture of 3a (0.14 g, 0.5 mmol), 4 (0.18 g, 0.6 mmol) andpiperidine (0.1 mL) in ethanol (40 mL) was heated to reflux overnight.After cooling down to room temperature, the organic solvent was removed.The residue was purified by recrystallization from methanol to affordSLM (0.24 g) as red solid in 56% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 9.28(d, J=6.4 Hz, 1H), 9.14 (d, J=8.4 Hz, 1H), 8.86 (s, 1H), 8.51 (d, J=6.4Hz, 1H), 8.42 (m, 3H), 8.28 (m, 2H), 8.13 (d, J=8.8 Hz, 1H), 8.08 (t,J=7.2 Hz, 1H), 7.80 (d, J=8.8 Hz, 1H), 7.71 (d, J=8.0 Hz, 1H), 7.53 (t,J=8.0 Hz, 1H), 7.32 (t, J=7.2 Hz, 1H), 4.64 (t, J=5.2 Hz, 2H), 4.52 (s,3H), 3.84 (t, J=5.2 Hz, 2H), 3.48 (m, 2H), 3.33 (m, 2H), 3.11 (s, 3H).¹³C NMR (400 MHz, DMSO-d₆) δ 153.0, 147, 144.9, 142.1, 140.9, 138.8,134.9, 129.0, 127.3, 126.7, 126.4, 126.1, 122.8, 122.2, 121.7, 120.4,119.9, 119.3, 116.2, 115.1, 110.5, 110.4, 71.3, 69.8, 68.9, 58.1, 44.2,42.9. HRMS (MALDI-TOF) m/z Calcd for C₂₉H₂₉N₂O₂ 437.2223. Found 437.2207[M⁺].

(E)-1-(2-hydroxyethyl)-4-(2-(9-(2-(2-methoxyethoxy)ethyl)-9H-carbazol-3-yl)vinyl)-quinoliniumchloride (SLOH)

A solution mixture of 3a (0.12 g, 0.55 mmol), 5 (0.2 g, 0.66 mmol) andpiperidine (0.1 mL) in ethanol (35 mL) was heated to reflux overnight.After cooling down to room temperature, the organic solvent was removed.The residue was purified by recrystallization from methanol to affordSLOH (0.17 g) as red solid in 62% yield. NMR (400 MHz, DMSO-d₆) δ 9.20(d, J=6.4 Hz, 1H), 9.15 (d, J=8.8 Hz, 1H), 8.87 (s, 1H), 8.56 (d, J=9.2Hz, 1H), 8.52 (d, J=6.4 Hz, 1H), 8.40 (m, 2H), 8.24 (m, 2H), 8.13 (d,J=8.8 Hz, 1H), 8.05 (t, J=7.6 Hz, 1H), 7.78 (d, J=8.8 Hz, 1H), 7.71 (d,J=8.4 Hz, 1H), 7.52 (t, J=8.0 Hz, 1H), 7.31 (t, J=7.6 Hz, 1H), 5.27 (t,J=5.6 Hz, 1H), 5.05 (t, J=4.8 Hz, 2H), 4.64 (t, J=4.8 Hz, 2H), 3.94 (m,2H), 3.84 (t, J=5.2 Hz, 2H), 3.47 (m, 2H), 3.31 (m, 2H), 3.11 (s, 3H).¹³C NMR (400 MHz, DMSO-d₆) δ 153.3, 147.8, 145.0, 142.1, 140.9, 138.1,134.7, 128.7, 127.1, 126.8, 126.7, 126.5, 122.8, 122.2, 121.7, 120.3,119.8, 119.2, 116.3, 114.8, 110.4, 110.3, 71.2, 69.8, 68.8, 58.9, 58.5,58.0, 42.9. HRMS (MALDI-TOF) m/z Calcd for C₃₀H₃₁N₂O₃ 467.2342. Found467.2340 [M⁺]. Calcd for C₃₀H₃₁ClN₂O₃: C, 71.53; H, 6.21; N, 5.57.Found: C, 71.04; H, 6.23; N, 5.36.

(E)-1-ethyl-4-(2-(9-(2-(2-methoxyethoxy)ethyl)-9H-carbazol-3-yl)vinyl)quinoliniumbromide (SLE)

A solution mixture of 6 (0.20 g, 0.8 mmol), 3a (0.33 g, 1.1 mmol) andpiperidine (0.1 mL) in ethanol (40 mL) was heated to reflux overnight.After cooling down to room temperature, the organic solvent was removed.The residue was purified by precipitation from methanol and ethylacetate to afford SLE (0.22 g) in 53% yield. ¹H NMR (400 MHz, DMSO-d₆) δ9.34 (d, J=8.4 Hz, 1H), 9.15 (d, J=8.4 Hz, 1H), 8.86 (s, 1H), 8.54-8.51(m, 2H), 8.44 (d, J=16 Hz, 1H), 8.36 (d, J=16 Hz, 1H), 8.28-8.23 (m,2H), 8.12 (d, J=8.0 Hz, 1H), 8.05 (t, J=7.6 Hz, 1H), 7.77 (d, J=8.4 Hz,1H), 7.70 (d, J=8.4 Hz, 1H), 7.52 (t, J=7.6 Hz, 1H), 7.31 (t, J=7.6 Hz,1H), 4.99 (tr, J=6.8 Hz, 2H), 4.63 (t, J=4.8 Hz, 2H), 3.84 (t, J=4.8 Hz,2H), 3.48 (t, J=4.8 Hz, 2H), 3.31 (t, J=4.8 Hz, 2H), 3.11 (s, 3H), 1.59(t, J=6.8 Hz, 3H). ¹³C NMR (400 MHz, DMSO-d₆) δ 153.2, 146.7, 145.1,142.2, 140.9, 137.7, 135.0, 128.9, 127.4, 126.8, 126.7, 126.5, 126.4,122.8, 122.2, 121.8, 120.4, 119.9, 119.0, 116.2, 115.5, 110.4, 110.3,71.3, 69.8, 68.9, 58.1, 51.9, 15.1. HRMS (MALDI-TOF) m/z Calcd forC₃₀H₃₁N₂O₂ 451.2380. Found 451.2362 [M]⁺.

(E)-1-(3-hydroxypropyl)-4-(2-(9-(2-(2-methoxyethoxy)ethyl)-9H-carbazol-3-yl)vinyl)-quinoliniumbromide (SLOH-Pr)

A solution mixture of 7 (0.17 g, 0.6 mmol), 3a (0.24 g, 0.8 mmol) andpiperidine (0.1 mL) in ethanol (40 mL) was heated to reflux overnight.After cooling down to room temperature, the organic solvent was removed.The residue was purified by precipitation from methanol and ethylacetate to afford SLOH-Pr (0.14 g) in 41% yield. ¹H NMR (400 MHz,DMSO-d₆) δ 9.29 (d, J=6.8 Hz, 1H), 9.15 (d, J=8.4 Hz, 1H), 8.88 (s, 1H),8.51 (d, J=6.8 Hz, 1H), 8.45 (d, J=16 Hz, 1H), 8.37 (d, J=16 Hz, 1H),8.28-8.24 (m, 2H), 8.13 (d, J=8.4 Hz, 1H), 8.05 (t, J=8.0 Hz, 1H), 7.77(d, J=8.8 Hz, 1H), 7.70 (d, J=8.4 Hz, 1H), 7.52 (t, J=7.6 Hz, 1H), 7.31(t, J=7.2 Hz, 1H), 5.01 (t, J=7.2 Hz, 2H), 4.86 (t, J=5.2 Hz, 1H), 4.63(t, J=4.8 Hz, 2H), 3.84 (t, J=5.2 Hz, 2H), 3.55 (tr, J=5.2 Hz, 2H), 3.47(t, J=5.6 Hz, 2H), 3.31 (t, J=4.8 Hz, 2H), 3.11 (s, 3H), 2.13 (t, J=6.0Hz, 2H). ¹³C NMR (400 MHz, DMSO-d₆) δ 153.3, 147.3, 145.1, 142.2, 140.9,137.9, 135.0, 128.8, 127.4, 126.8, 126.7, 126.5, 126.4, 122.8, 122.2,121.8, 120.4, 119.9, 119.0, 116.3, 115.2, 110.5, 110.4, 71.3, 69.8,68.9, 58.1, 57.6, 54.2, 42.9, 32.0. HRMS (MALDI-TOF) m/z Calcd forC₃₁H₃₃N₂O₃ 481.2485. Found 481.2458 [M]⁺.

(E)-1-methyl-4-(2-(9-methyl-9H-carbazol-3-yl)vinyl)quinolinium iodide(Me-SLM)

A solution mixture of 1,4-dimethylquinolinium iodide (0.14 g, 0.5 mmol),3b (0.13 g, 0.6 mmol) and piperidine (0.1 mL) in ethanol (40 mL) washeated to reflux overnight. After cooling down to room temperature, theorganic solvent was removed. The residue was purified by precipitationfrom methanol and ethyl acetate to afford Me-SLM (0.14 g) in 62% yield.¹H NMR (400 MHz, DMSO-d₆) δ 9.27 (d, J=6.4 Hz, 1H), 9.12 (d, J=8.4 Hz,1H), 8.86 (s, 1H), 8.49 (d, J=6.4 Hz, 1H), 8.45-8.23 (m, 5H), 8.15 (d,J=8.8 Hz, 1H), 8.06 (t, J=7.6 Hz, 1H), 7.75 (d, J=8.4 Hz, 1H), 7.66 (d,J=8.0 Hz, 1H), 7.55 (t, J=7.6 Hz, 1H), 7.32 (t, J=7.6 Hz, 1H), 4.51 (s,3H), 3.95 (s, 3H). ¹³C NMR (400 MHz, DMSO-d₆) δ 152.9, 147.3, 144.8,142.2, 141.2, 138.7, 134.8, 128.8, 127.4, 126.6, 126.4, 126.3, 126.0,122.6, 122.0, 121.8, 120.4, 119.7, 119.1, 116.0, 115.0, 109.8, 109.7,44.3, 29.3. HRMS (MALDI-TOF) m/z Calcd for C₂₅H₂₁N₂ 349.1699. Found349.1694 [M]⁺.

9-(bromomethyl)acridine (10)

To a solution of 9-methylacridine (1.93 g, 10 mmol) in dichloromethane(100 mL) was added NBS (1.78 g, 10 mmol) portion-wise in an ice-waterbath. After complete addition, the solution mixture was warmed to roomtemperature and stirred overnight. The resulting solution was washedwith water and brine. The organic phase was dried over anhydrous sodiumsulfate and the solvent was removed. The residue was purified by silicagel chromatography using ethyl acetate and petroleum ether (EA:PE=1:5)as eluent to afford 10 (2.08 g) in 77% yield. ¹H NMR (400 MHz, CDCl₃) δ8.27 (d, J=8.8 Hz, 4H), 7.81 (t, J=8.0 Hz, 2H), 7.68 (t, J=8.0 Hz, 2H),5.42 (s, 2H). ¹³C NMR (400 MHz, CDCl₃) δ 148.9, 138.7, 130.5, 130.1,126.8, 123.8, 123.4, 23.1. MS (FAB) m/z Calcd for C₁₄H₁₀BrN 272.1. Found2722. [M]⁺.

Diethyl acridin-9-ylmethylphosphonate (11)

The mixture of 10 (1.5 g, 5.5 mmol) and triethyl phosphite (2 mL) washeated to reflux for 4 h. After cooling down to room temperature, theexcess triethyl phosphite was removed under vacuum to afford 11 (1.7 g)in 94% yield. NMR (400 MHz, CDCl₃) δ 8.23 (d, J=8.8 Hz, 2H), 8.17 (d,J=8.8 Hz, 2H), 7.72 (t, J=7.2 Hz, 2H), 7.54 (t, J=7.2 Hz, 2H), 4.13 (d,J=24 Hz, 2H), 3.92-3.77 (m, 4H), 1.04 (t, J=7.2 Hz, 6H). ¹³C NMR (400MHz, CDCl₃) δ 148.4, 148.3, 135.8, 135.7, 129.9, 129.8, 125.8, 125.3,125.2, 124.9, 124.8, 62.4, 27.5, 26.1, 16.1.

(E)-9-(2-(9-(2-(2-methoxyethoxy)ethyl)-9H-carbazol-3-yl)vinyl)acridine(12)

To a solution of 3a (0.45 g, 1.5 mmol) and 11 (0.49 g, 1.5 mmol) in dryTHF (45 mL), NaH (45 mg, 1.8 mmol) was added carefully in an ice-waterbath. After complete addition, the solution mixture was warmed to roomtemperature and stirred overnight. After quenching by water, theresulting mixture was extracted with ethyl acetate for three times. Thecombined organic phase was washed with brine twice and dried overanhydrous sodium sulfate. After removing the solvent, the resultingcrude product was purified by silica gel chromatography using DCM andpetroleum ether (DCM:PE=1:10) to afford 12 (0.45 g) in 64% yield. ¹H NMR(400 MHz, CDCl₃) δ 8.45 (d, J=8.8 Hz, 2H), 8.37 (s, 1H), 8.26 (d, J=8.8Hz, 2H), 8.15 (d, J=8.0 Hz, 1H), 7.95 (d, J=8.4 Hz, 1H), 7.85 (d, J=8.8Hz, 1H), 7.80 (t, J=8.0 Hz, 2H), 7.58-7.51 (m, 5H), 7.31-7.25 (m, 2H),4.58 (t, J=6.4 Hz, 2H), 3.92 (t, J=6.4 Hz, 2H), 3.57-3.55 (m, 2H),3.48-3.45 (m, 2H), 3.35 (s, 3H). ¹³C NMR (400 MHz, CDCl₃) δ 148.9,143.8, 141.0, 140.6, 129.9, 127.9, 126.1, 125.4, 124.6, 123.4, 122.9,120.4, 119.5, 119.2, 119.1, 109.4, 109.2, 71.9, 70.9, 69.3, 59.1, 43.3.HRMS (MALDI-TOF) m/z Calcd for C₃₂H₂₉N₂O₂ 473.2223. Found 473.2210[M+H]⁺.

(E)-9-(2-(9-(2-(2-methoxyethoxy)ethyl)-9H-carbazol-3-yl)vinyl)-10-methylacridiniumiodide (SAM)

A solution of 12 (0.20 g, 0.4 mmol) and methyl iodide (0.57 g, 4 mmol)in acetonitrile (8 mL) was heated to 100° C. in sealed tube for 24 h.After cooling down to room temperature, the solvent was removed and theresulting mixture was purified by precipitation from methanol and ethylacetate to afford SAM (0.15 g) in 61% yield. ¹H NMR (400 MHz, CDCl₃) δ8.74 (d, J=8.0 Hz, 2H), 8.49 (s, 1H), 8.46 (d, J=8.8 Hz, 2H), 8.31 (d,J=16 Hz, 1H), 8.26 (t, J=8.0 Hz, 2H), 8.10 (d, J=8.0 Hz, 1H), 7.93 (d,J=8.0 Hz, 1H), 7.83 (t, J=7.2 Hz, 211), 7.51 (d, J=8.4 Hz, 1H), 7.46 (t,J=6.4 Hz, 2H), 7.44 (d, J=16 Hz, 1H), 7.20 (t, J=6.4 Hz, 1H), 4.82 (s,3H), 4.46 (t, J=6.0 Hz, 2H), 3.88 (t, J=6.0 Hz, 2H), 3.55-3.53 (m, 2H),3.44-3.42 (m, 2H), 3.30 (s, 3H). ¹³C NMR (400 MHz, CDCl₃) δ 157.9,149.5, 141.8, 140.5, 140.1, 137.8, 129.3, 127.4, 126.9, 126.5, 126.2,123.9, 123.2, 122.1, 121.2, 121.1, 119.9, 117.9, 117.4, 109.4, 109.0,71.7, 70.6, 69.0, 58.9, 43.3, 39.5. HRMS (MALDI-TOF) m/z Calcd forC₃₃H₃₁N₂O₂ ⁺487.2380. Found 487.2387 [M]⁺.

(E)-10-(2-hydroxyethyl)-9-(2-(9-(2-(2-methoxyethoxy)ethyl)-9H-carbazol-3-yl)vinyl)-acridiniumiodide (SAOH)

A solution of 12 (0.2 g, 0.4 mmol) and 2-iodoethanol (0.7 g, 4 mmol) inacetonitrile (10 mL) was heated to 120° C. in sealed tube for 24 h.After cooling down to room temperature, the solvent was removed and theresulting mixture was purified by precipitation from methanol and ethylacetate to afford SAOH (0.13 g) in 52% yield. ¹H NMR (400 MHz, CDCl₃) δ8.98 (d, J=9.2 Hz, 2H), 8.77 (d, J=8.4 Hz, 2H), 8.44 (s, 1H), 8.36 (t,J=8.0 Hz, 2H), 8.18 (d, J=8.4 Hz, 1H), 8.17 (d, J=16 Hz, 1H), 7.91 (d,J=8.0 Hz, 1H), 7.86 (t, J=8.0 Hz, 2H), 7.62 (d, J=8.8 Hz, 1H), 7.55 (d,J=8.4 Hz, 1H), 7.52 (t, J=6.4 Hz, 2H), 7.48 (d, J=16 Hz, 1H), 7.33 (t,J=6.4 Hz, 1H), 5.63 (t, J=6.0 Hz, 2H), 4.75 (t, J=7.6 Hz, 1H), 4.59 (t,J=6.0 Hz, 2H), 4.51-4.47 (m, 2H), 3.94 (t, J=6.0 Hz, 2H), 3.57-3.55 (m,2H), 3.47-3.44 (m, 2H), 3.33 (s, 3H). ¹³C NMR (400 MHz, CDCl₃) δ 158.2,149.1, 141.9, 140.7, 140.6, 138.1, 129.1, 126.9, 126.5, 126.4, 124.3,123.3, 122.4, 121.2, 120.9, 120.0, 119.0, 117.0, 109.6, 109.2, 71.8,70.6, 69.1, 59.3, 58.9, 52.2, 43.3. HRMS (MALDI-TOF) m/z Calcd forC₃₄H₃₃N₂O₃ ⁺517.2486. Found 517.2476 [M]⁺.

While the foregoing invention has been described in terms of theembodiments discussed above, numerous variations are possible.Accordingly, modifications and changes such as those suggested above,but not limited thereto, are considered to be within the scope offollowing claims.

What is claimed is:
 1. A method for treating beta-amyloid (Aβ) peptidesaggregation-associated diseases and preventing development andprogression of said diseases due to increased Aβ aggregates-inducedneurotoxicity by administering carbazole-based fluorophores comprisingformula S or V series,

said method comprising: binding said carbazole-based fluorophores to Aβpeptides, oligomers and fibrils thereof; inhibiting the growth and/oraggregation of said Aβ peptides, oligomers and/or fibrils upon saidbinding; and protecting neuronal cells against the neurotoxic activitiesof the Aβ oligomers and/or fibrils; wherein in said carbazole-basedfluorophores, Ar is a heteraromatic ring selected from the groupconsisting of pyridinyl, substituted pyridinyl, quinolinyl, substitutedquinolinyl, acridinyl, substituted acridinyl, benzothiazolyl,substituted benzothiazolyl, benzoxazolyl, and substituted benzoxazolyl;R₁ is selected from the group consisting of polyethylene glycol chain,alkyl, substituted alkyl, peptide chain, glycosidyl, andC(O)NHCH((CH₂CH₂O)₂CH₃)₂; R₂ is selected from the group consisting ofethenyl, ethynyl, azo and azomethinyl; R₃ is selected from the groupconsisting of alkyl, HO-alkyl, HS-alkyl, H₂N-alkyl, HNalkyl-alkyl,HOOC-alkyl, (alkyl)₃N⁺-alkyl, and (Ph)₃P⁺-alkyl; and X is selected fromthe group consisting of F⁻, Cl⁻, Br⁻, I⁻, HSO₄ ⁻, H₂PO₄ ⁻, HCO₃ ⁻,tosylate, and mesylate.
 2. The method according to claim 1 wherein saidAr is selected from a quinolinyl or substituted quinolinyl; said R₁ is a2-(2-methoxyethoxyl)ethoxy; said R₂ is an ethenyl; said R₃ is selectedfrom a methyl, 2-hydroxyethyl or ethyl or 3-hydroxypropyl; and said X isselected from a chloride, bromide or iodide, and the carbazole-basedfluorophores of which are represented by the formula SLM, SLOH, SLE andSLOH-Pr:


3. The method according to claim 1, wherein said Ar is selected from aquinolinyl or substituted quinolinyl; said R₁ is a methyl; said R₂ is anethenyl; said R₃ is a methyl; and said X is selected from a chloride,bromide or iodide, and the carbazole-based fluorophores of which arerepresented by the formula Me-SLM:


4. The method according to claim 1, wherein said Ar is selected from anacridinyl or substituted acridinyl; said R₁ is a2-(2-methoxy-ethoxy)ethoxy; said R₂ is an ethenyl; said R₃ is selectedfrom a methyl or 2-hydroxyethyl; and said X is selected from a chloride,bromide or iodide, and the carbazole-based fluorophores of which arerepresented by the formula SAM and SAOH:


5. The method according to claim 1, wherein said Ar is selected from apyridinyl or substituted pyridinyl; said R₁ is a2-(2-methoxyethoxyl)ethoxy; said R₂ is an ethenyl; said R₃ is selectedfrom a methyl or 2-hydroxyethyl; and said X is selected from a chloride,bromide or iodide, and the carbazole-based fluorophores of which arerepresented by the formula SPM and SPOH:


6. The method according to claim 1, wherein the carbazole-basedfluorophores are water-soluble.
 7. The method according to claim 1,wherein the carbazole-based fluorophores are non-toxic.
 8. The methodaccording to claim 1, wherein the carbazole-based fluorophores are ableto pass through the blood-brain barrier.
 9. The method according toclaim 1, wherein the carbazole-based fluorophores are carbazole-basedcyanines.
 10. The method according to claim 1, wherein thecarbazole-based fluorophores are administered in vitro, in vivo, or invitro and in vivo.
 11. The method according to claim 1, wherein thebeta-amyloid (Aβ) peptides aggregation-associated diseases includeAlzheimer's disease.